TY - JOUR
T1 - Acidic residues in the purine binding site govern the 6-oxopurine specificity of the Leishmania donovani xanthine phosphoribosyltransferase
AU - Ullman, Buddy
AU - Cyr, Normand
AU - Choi, Kenneth
AU - Jardim, Armando
N1 - Funding Information:
This work was supported by a grant from Natural Science and Engineering Research Council of Canada (A.J.), National Institute of Allergy and Infectious Diseases Grant AI23682 (B.U.), and Fonds Québécois de la Recherche sur la Nature et les Technologies (FQRNT) Regroupement Stratégique . N.C. is supported by a doctoral scholarship from FQRNT. We thank Dr. Joanne Turnbull of Concordia University for assistance with the circular dichroism experiments.
PY - 2010/2
Y1 - 2010/2
N2 - Leishmania possess distinct xanthine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase enzymes that mediate purine salvage, an obligatory nutritional function for these pathogenic parasites. The xanthine phosphoribosyltransferase preferentially uses xanthine as a substrate, while the hypoxanthine-guanine phosphoribosyltransferase phosphoribosylates only hypoxanthine and guanine. These related phosphoribosyltransferases were used as model system to investigate the molecular determinants regulating the 6-oxopurine specificity of these enzymes. Analysis of the purine binding domains showed two conserved acidic amino acids; glutamate residues in the xanthine phosphoribosyltransferase (E198 and E215) and aspartate residues in the hypoxanthine-guanine phosphoribosyltransferase (D168 and D185). Genetic and biochemical analysis established that the single E198D and E215D mutations increased the turnover rates of the xanthine phosphoribosyltransferase without altering purine nucleobase specificity. However, the E215Q and E198,215D mutations converted the Leishmania xanthine phosphoribosyltransferase into a broad-specificity enzyme capable of utilizing guanine, hypoxanthine, and xanthine as substrates. Similarly, the D168,185E double mutation transformed the Leishmania hypoxanthine-guanine phosphoribosyltransferase into a mutant enzyme capable phosphoribosylating only xanthine, albeit with a much lower catalytic efficiency. These studies established that these conserved acidic residues play an important role in governing the nucleobase selectivity of the Leishmania 6-oxopurine phosphoribosyltransferases.
AB - Leishmania possess distinct xanthine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase enzymes that mediate purine salvage, an obligatory nutritional function for these pathogenic parasites. The xanthine phosphoribosyltransferase preferentially uses xanthine as a substrate, while the hypoxanthine-guanine phosphoribosyltransferase phosphoribosylates only hypoxanthine and guanine. These related phosphoribosyltransferases were used as model system to investigate the molecular determinants regulating the 6-oxopurine specificity of these enzymes. Analysis of the purine binding domains showed two conserved acidic amino acids; glutamate residues in the xanthine phosphoribosyltransferase (E198 and E215) and aspartate residues in the hypoxanthine-guanine phosphoribosyltransferase (D168 and D185). Genetic and biochemical analysis established that the single E198D and E215D mutations increased the turnover rates of the xanthine phosphoribosyltransferase without altering purine nucleobase specificity. However, the E215Q and E198,215D mutations converted the Leishmania xanthine phosphoribosyltransferase into a broad-specificity enzyme capable of utilizing guanine, hypoxanthine, and xanthine as substrates. Similarly, the D168,185E double mutation transformed the Leishmania hypoxanthine-guanine phosphoribosyltransferase into a mutant enzyme capable phosphoribosylating only xanthine, albeit with a much lower catalytic efficiency. These studies established that these conserved acidic residues play an important role in governing the nucleobase selectivity of the Leishmania 6-oxopurine phosphoribosyltransferases.
KW - HGPRT
KW - Leishmania
KW - Phosphoribosyltransferase
KW - Purine metabolism
KW - XPRT
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U2 - 10.1016/j.biocel.2009.10.020
DO - 10.1016/j.biocel.2009.10.020
M3 - Article
C2 - 19861168
AN - SCOPUS:73749085906
SN - 1357-2725
VL - 42
SP - 253
EP - 262
JO - International Journal of Biochemistry and Cell Biology
JF - International Journal of Biochemistry and Cell Biology
IS - 2
ER -